The sodium chloride co-transporter (NCC) and the epithelial sodium channel (ENaC) are two key sodium transporting proteins in the distal tubule of the mammalian kidney. Hyperactivity of either protein results in hypertension and hypoactivity results in hypotension. These two proteins are co-expressed in the second part of the distal convoluted tubule (DCT2) and are regulated by many of the same proteins. However, associations between the two have not been investigated. Preliminary data indicates that the proteins associate in a sodium transporting complex and co-localize in the apical membrane of the DCT2. Investigating the relationship between ENaC and NCC in this key nephron segment has the potential to significantly alter our understanding of salt transport in the mammalian kidney. We now propose to examine the interaction of NCC with each subunit of ENaC, examine the functional implications of this interaction and investigate association-dependent alterations in regulation of these proteins by aldosterone. Co-localization (in vivo and in vitro), native association, and proximity of NCC and each ENaC subunit will be examined. The ENaC subunit(s) that are critical for binding to NCC will also be determined. These studies will define the association between NCC and each ENaC subunit. Then the impact of this association on function, protein expression, surface expression and response to inhibitors of both sodium transporters will be investigated. These studies will determine the functional implications of this interaction. Then aldosterone-induced changes in function, protein expression and surface expression of NCC and ENaC will be examined in the absence or presence of the other. The aldosterone-induced phosphorylation of NCC with and without ENaC will also be investigated. Additionally the association of NEDD4-2 with NCC in the absence and presence of ENaC subunits will be determined. These studies will determine whether the NCC alters the regulation of ENaC by aldosterone and whether any of the ENaC subunits alter the regulation of NCC by aldosterone. A variety of methods will be utilized to address these issues. Function of NCC will be assessed by thiazide-sensitive 22Na+ uptakes in mouse distal convoluted tubule cells (mDCT15). For measurement of ENaC function, Dr. Doug Eaton's lab will use single channel whole-cell patch clamping to assess ENaC function. The proximity of these proteins will be assessed utilizing Fluorescence Resonance Energy Transfer (FRET). Signal will be detected with confocal microscopy. Native association will be examined through the use of Blue Native PAGE. Electron Microscopy will be used to examine association under aldosterone stimulation. Lentiviral shRNA transduction particles will be used to knockdown protein expression in mDCT15 cells. Small animal surgeries with implantation of osmotic minipumps will be used to examine the effects of aldosterone on the NCC-ENaC association on the organismal level. Additionally standard methodologies such as immunoblotting, SDS-PAGE, biotinylation, transfection, co- immunoprecipitation and immunohistochemistry will be used.